Phosphonate compounds

ABSTRACT

The present invention relates to phosphonate compounds, compositions containing them, processes for obtaining them, and their use for treating a variety of medical disorders, e.g., osteoporosis and other disorders of bone metabolism, cancer, viral infections, and the like.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/645,105 filed Oct. 4, 2012, now pending; which is a continuationapplication of U.S. application Ser. No. 13/220,548 filed Aug. 29, 2011,now issued as U.S. Pat. No. 8,309,565; which is a continuationapplication of U.S. application Ser. No. 12/701,410 filed Feb. 5, 2010,now issued as U.S. Pat. No. 8,008,308; which is a continuationapplication of U.S. application Ser. No. 11/925,309 filed Oct. 26, 2007,now issued as U.S. Pat. No. 7,687,480; which is a continuationapplication of U.S. application Ser. No. 11/715,604 filed Mar. 7, 2007,now issued as U.S. Pat. No. 7,790,703; which is a continuationapplication of U.S. application Ser. No. 11/506,292 filed Aug. 17, 2006,now issued as U.S. Pat. No. 7,452,898; which is a continuationapplication of U.S. application Ser. No. 11/100,882 filed Apr. 6, 2005,now issued as U.S. Pat. No. 7,094,772; which is a continuationapplication of U.S. application Ser. No. 10/759,345 filed Jan. 15, 2004,now issued as U.S. Pat. No. 7,034,014; which is a continuationapplication of U.S. application Ser. No. 10/148,374 filed Nov. 6, 2002,now issued as U.S. Pat. No. 6,716,825; which is a 35 USC §371 NationalStage application of International Application No. PCT/US00/33079 filedDec. 4, 2000, which claims the benefit under 35 USC §119(e) to U.S.Application Ser. No. 60/205,719 filed May 19, 2000, and to U.S.Application Ser. No. 60/168,813 filed Dec. 3, 1999. The disclosure ofeach of the prior applications is considered part of and is incorporatedby reference in the disclosure of this application.

GRANT INFORMATION

This invention was made with government support under Grant No. GM24979awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel phosphonate compounds,compositions containing them, processes for producing them, and theiruse for treating a variety of medical disorders, e.g., osteoporosis andother disorders of bone metabolism, cancer, viral infections, and thelike.

2. Background Information

Phosphonate compounds have long been known to provide a variety oftherapeutic benefits. A particular class of therapeutically beneficialphosphonate compounds are the bisphosphonates, i.e., pyrophosphateanalogs wherein the central oxygen atom of the pyrophosphate bond isreplaced by carbon. Various substituent groups may be attached to thiscentral carbon atom to produce derivative bisphosphonate compoundshaving various degrees of pharmacological potency. These derivativeshave the general structure:

wherein R_(a) and R_(b) may independently be selected from hydroxyl,amino, sulfhydryl, halogen, or a variety of alkyl or aryl groups, or acombination of such groups, which may be further substituted. Examplesinclude Etidronate, wherein R_(a) is CH₃ and R_(b) is OH; Clodronate,dichloromethylene bisphosphonic acid (CI₂MDP), wherein R_(a) and R_(b)are Cl, Pamidronate, 3-amino-1-hydroxypropylidene bisphosphonic acid,wherein R_(a) is ethylamino and R_(b) is hydroxyl; Alendronate,4-amino-1-hydroxybutylidene bisphosphonic acid, wherein R_(a) ispropylamino and R_(b) is hydroxyl; Olpadronate,3-dimethylamino-1-hydroxypropylidene bisphosphonic acid, wherein R_(a)is dimethylaminoethyl and R_(b) is hydroxyl; and amino-olpadronate(IG-9402), 3-(N,N-dimethylamino)-1-aminopropylidene bisphosphonate,wherein R_(a) is N,N-dimethylaminoethyl and R_(b) is NH2.

Bisphosphonates and their substituted derivatives have the intrinsicproperty of inhibiting bone resorption in vivo. Bisphosphonates also actby inhibiting apoptosis (programmed cell death) in bone-forming cells.Indications for their use therefore include the treatment and preventionof osteoporosis, treatment of Paget's disease, metastatic bone cancers,hyperparathyroidism, rheumatoid arthritis, algodistrophy,stemo-costo-clavicular hyperostosis, Gaucher's disease, Engleman'sdisease, and certain non-skeletal disorders. (Papapoulos, S. E., inOsteoporosis, R. Marcus, D. Feldman and J. Kelsey, eds., Academic Press,San Diego, 1996. p. 1210, Table 1).

Although bisphosphonates have therapeutically beneficial properties,they suffer from pharmacological disadvantages as orally administeredagents. One drawback is low oral availability: as little as 0.7% to 5%of an orally administered dose is absorbed from the gastrointestinaltract. Oral absorption is further reduced when taken with food. Further,it is known that some currently available bisphosphonates, e.g.,FOSAMAX™ (Merck; alendronate sodium), SKELID™ (Sanofi, tiludronate) andACTONE™ (Procter and Gamble, risedronate) have local toxicity, causingesophageal irritation and ulceration. Other bisphosphonates, likeamino-olpadronate, lack anti-resorptive effects (Van Beek, E. et al., J.Bone Miner Res 11(10):1492-1497 (1996) but inhibit osteocyte apoptosisand are able to stimulate net bone formation (Plotkin, L. et al., J ClinInvest 104(10): 1363-1374 (1999) and U.S. Pat. No. 5,885,973). It wouldtherefore, be useful to develop chemically modified bisphosphonatederivatives that maintain or enhance the pharmacological activity of theparent compounds while eliminating or reducing their undesirable sideeffects.

In addition to bisphosphonates, monophosphonates are also known toprovide therapeutic benefits. One class of therapeutically beneficialmonophosphonates are the antiviral nucleotide phosphonates, such as, forexample, cidofovir, cyclic cidofovir, adefovir, tenofovir, and the like,as well as the 5′-phosphonates and methylene phosphonates ofazidothymidine, ganciclovir, acyclovir, and the like. In compounds ofthis type, the 5′-hydroxyl of the sugar moiety, or its equivalent inacyclic nucleosides (ganciclovir, penciclovir, acyclovir) which do notcontain a complete sugar moiety, is replaced with a phosphorus-carbonbond. In the case of the methylene phosphonates, a methylene groupreplaces the 5′-hydroxyl or its equivalent, and its carbon atom is, inturn, covalently linked to the phosphonate. Various AZT structures arepresented below, including compounds contemplated for use in thepractice of the present invention. AZT itself is shown on the left.Compound A is AZT-monophosphate which has the usual phosphodiester linkbetween the sugar and the phosphate. In contrast, in compounds B (AZT5′-phosphonate) and C (AZT 5′-methylene phosphonate), the 5′-hydroxyl of3′-azido, 2′,3′-dideoxyribose is absent and has been replaced by eithera phosphorus-carbon bond (AZT phosphonate) or by a methylene linked by aphosphorus-carbon bond (AZT methylene phosphonate). Compounds B and Care examples of compounds useful in the practice of the presentinvention.

Compounds of this type may be active as antiproliferative or antiviralnucleotides. Upon cellular metabolism, two additional phosphorylationsoccur to form the nucleotide phosphonate diphosphate which representsthe equivalent of nucleoside triphosphates. Antiviral nucleotidephosphonate diphosphates are selective inhibitors of viral RNA or DNApolymerases or reverse transcriptases. That is to say, their inhibitoryaction on viral polymerases is much greater than their degree ofinhibition of mammalian cell DNA polymerases α, β and γ or mammalian RNApolymerases. Conversely, the antiproliferative nucleotide phosphonatediphosphates inhibit cancer cell DNA and RNA polymerases and may showmuch lower selectivity versus normal cellular DNA and RNA polymerases.Since nucleotide phosphonates are poorly absorbed from the GI tract theyfrequently require parenteral administration (e.g. cidofovir).Furthermore, the negatively charged phosphonate moiety may interferewith cellular penetration, resulting in reduced activity as antiviralsor antiproliferatives. Invention compounds may surprisingly overcome thedisadvantages of this class of agents.

Pharmacologically active agents of antiviral phosphonates are known; thefollowing U.S. patents describe other approaches for nucleotidephosphonate analogs: U.S. Pat. No. 5,672,697 (Nuleoside-5′-methylenephosphonates), U.S. Pat. No. 5,922,695 (Antiviral phosphonomethoxynucleotide analogs), U.S. Pat. No. 5,977,089 (Antiviral phosphonomethoxynucleotide analogs), U.S. Pat. No. 6,043,230 (Antiviral phosphonomethoxynucleotide analogs), U.S. Pat. No. 6,069,249. The preparation and use ofalkylglycerol phosphates covalently linked to non-phosphonate containingdrugs having amino, carboxyl, hydroxyl or sulfhydryl functional groupshave previously been disclosed. These prodrugs optionally comprise alinker group or one or two additional phosphates esters between the drugand the alkyl glycerol phosphate (U.S. Pat. No. 5,411,947 and U.S.patent application Ser. No. 08/487,081). Partial esters ofchloromethanediphosphonic acid are known (U.S. Pat. No. 5,376,649) anddianhydrides of clodronate have been reported (Ahlniark, et al., J MedChem 42: 1473-1476 (1999)). However, the partial esters were found tonot release the active bisphosphonate by chemical or biochemicalconversion (Niemi, R. et al, J Chrom B 701:97-102 (1997)). Prodrugscomprising alkylglycerol phosphate residues attached to antiviralnucleosides (U.S. Pat. No. 5,223,263) or phosphono-carboxylates (U.S.Pat. No. 5,463,092) have also been described.

There is, therefore, a continuing need for less toxic, more effectivepharmaceutical agents to treat a variety of disorders, such as thosecaused by viral infection and inappropriate cell proliferation, e.g.,cancer. Thus, it is an object of the present invention to developchemically modified phosphonate derivatives of pharmacologically activeagents, e.g., antiviral and anti-neoplastic pharmaceutical agents. Thesemodified derivatives increase the potency of the parent compound whileminimizing deleterious side effects when administered to a subject inneed thereof.

SUMMARY OF THE INVENTION

The invention provides analogs of phosphonate compounds. Phosphonatecompounds contemplated for use in accordance with the invention includethose that decrease bone resorption or inhibit osteoblast or osteocyteapoptosis, as well as those that improve the bioactivity, selectivity,or bioavailability of nucleotide phosphonate analogs which are usefulfor the treatment of cancer, various viral infections, and the like.Invention compounds comprise phosphonates covalently linked (directly orindirectly through a linker molecule) to a substituted or unsubstitutedalkylglycerol, alkylpropanediol, alkylethanediol, or related moiety. Inanother aspect of the present invention, there are providedpharmaceutical formulations containing the analogs of phosphonatecompounds described herein.

In accordance with another aspect of the present invention, there areprovided a variety of therapeutic methods, e.g., methods for treating orpreventing bone resorption in a mammal, methods for increasing boneformation by preventing osteoblast and osteocyte apoptosis, methods forincreasing bone mass and strength, methods for treating viralinfections, methods for treating disorders caused by inappropriate cellproliferation, e.g., cancer, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 summarizes the effect of a compound according to the invention,1-O-hexadecyloxypropane alendronate, on dexamethasone-induced apoptosisof MLO-Y4 osteocytic cells. Bars represent the mean±SD of 3 independentmeasurements. Open bars represent the absence of dexamethasone anddarkened bars represent the presence of 10⁻⁴ M dexamethasone.

FIG. 2 summarizes the effect of a compound according to the invention,1-O-hexadecyloxypropane alendronate, on dexamethasone-induced apoptosisof calvarial cells. Bars represent the mean±SD of 3 independentmeasurements. Gray bars represent the absence of dexamethasone and blackbars represent the presence of 10⁻⁴ M dexamethasone.

DETAILED DESCRIPTION OF THE INVENTION

The phosphonate compounds of the invention have the structure:

wherein:

R₁ and R₁′ are independently —H, optionally substituted —O(C₁-C₂₄)alkyl,—O(C₁-C₂₄)alkenyl, —O(C₁-C₂₄)acyl, —S(C₁-C₂₄)alkyl, —S(C₁-C₂₄)alkenyl,or —S(C₁-C₂₄)acyl, wherein at least one of R₁ and R₁′ are not —H, andwherein said alkenyl or acyl moieties optionally have 1 to 6 doublebonds,

R₂ and R₂′ are independently —H, optionally substituted —O(C₁-C₇)alkyl,—O(C₁-C₇)alkenyl, —S(C₁-C₇)alkyl, —S(C₁-C₇)alkenyl, —O(C₁-C₇)acyl,—S(C₁-C₇)acyl, —N(C₁-C₇)acyl, —NH(C₁-C₇)alkyl, —N((C₁-C₇)alkyl)₂, oxo,halogen, —NH₂, —OH, or —SH;

R₃ is a pharmaceutically active phosphonate, bisphosphonate or aphosphonate derivative of a pharmacologically active compound, linked toa functional group on optional linker L or to an available oxygen atomon C^(α);

X, when present, is:

L is a valence bond or a bifunctional linking molecule of the formula-J-(CR₂)₁-G-, wherein t is an integer from 1 to 24, J and G areindependently —O—, —S—, —C(O)O—, or —NH—, and R is —H, substituted orunsubstituted alkyl, or alkenyl;

m is an integer from 0 to 6; and

n is 0 or 1.

In preferred embodiments, m=0, 1 or 2. In these preferred embodiments,R₂ and R₂′ are preferably H, and the prodrugs are then ethanediol,propanediol or butanediol derivatives of a therapeutic phosphonate. Apreferred ethanediol phosphonate species has the structure

wherein R₁, R₁′, R₃, L, and n are as defined above.

A preferred propanediol species has the structure:

wherein m=1 and R₁, R₁′, R₃, L and n are as defined above in the generalformula.

A preferred glycerol species has the structure:

wherein m=1, R₂=H, R₂′=OH, and R₂ and R₂′ on C^(α) are both —H. Glycerolis an optically active molecule. Using the stereospecific numberingconvention for glycerol, the sn-3 position is the position which isphosphorylated by glycerol kinase. In compounds of the invention havinga glycerol residue, the -(L)_(n)-R₃ moiety may be joined at either thesn-3 or sn-1 position of glycerol.

In all species of the pharmacologically active agents of the invention,R₁ is preferably an alkoxy group having the formula —O—(CH₂)_(t)—CH₃,wherein t is 0-24. More preferably t is 11-19. Most preferably t is 15or 17.

Preferred R₃ groups include bisphosphonates that are known to beclinically useful, for example, the compounds:

-   Etidronate: 1-hydroxyethylidene bisphosphonic acid (EDHP);-   Clodronate: dichloromethylene bisphosphonic acid (CI2MDP);-   Tiludronate: chloro-4-phenylthiomethylene bisphosphonic acid;-   Pamidronate: 3-amino-1-hydroxypropylidene bisphosphonic acid (ADP);-   Alendronate: 4-amino-1-hydroxybutylidene bisphosphonic acid;-   Olpadronate: 3-dimethylamino-1-hydroxypropylidene bisphosphonic acid    (dimethyl-APD);-   Ibandronate: 3-methylpentylamino-1-hydroxypropylidene bisphosphonic    acid (BM 21.0955);-   EB-1053: 3-(1-pyrrolidinyl)-1-hydroxypropylidene bisphosphonic acid;-   Risedronate: 2-(3-pyridinyl)-1-hydroxy-ethylidene bisphosphonic    acid;-   Amino-Olpadronate:    3-(N,N-diimethylamino-1-aminopropylidene)bisphosphonate (IG9402),    and the like.

R₃ may also be selected from a variety of phosphonate-containingnucleotides (or nucleosides which can be derivatized to theircorresponding phosphonates), which are also contemplated for use herein.Preferred nucleosides include those useful for treating disorders causedby inappropriate cell proliferation such as 2-chloro-deoxyadenosine,1-β-D-arabinofuranosyl-cytidine (cytarabine, ara-C), fluorouridine,fluorodeoxyuridine (floxuridine), gemcitabine, cladribine, fludarabine,pentostatin (2′-deoxycoformycin), 6-mercaptopurine, 6-thioguanine, andsubstituted or unsubstituted 1-β-D-arabinofuranosyl-guanine (ara-G),1-β-D-arabinofuranosyl-adenosine (ara-A), 1-β-D-arabinofuranosyl-uridine(ara-U), and the like.

Nucleosides useful for treating viral infections may also be convertedto their corresponding 5′-phosphonates for use as an R₃ group. Suchphosphonate analogs typically contain either a phosphonate (—PO₃H₂) or amethylene phosphonate (—CH₂—PO₃H₂) group substituted for the 5′-hydroxylof an antiviral nucleoside. Some examples of antiviral phosphonatesderived by substituting —PO3H2 for the 5′-hydroxyl are:

3′-azido-3′,5′- dideoxythymidine-5′- phosphonic acid (AZT phosphonate)

Hakimelahi, G. H.; Moosavi-Movahedi, A. A.; Sadeghi, M. M.; Tsay, S-C.;Hwu, J. R., J. Med. Chem. 1995, 38: 4648-4659. 3′,5′-dideoxyhymidine-2′-ene-5′-phosphonic acid (d4T phosphonate)

″ 2′,3′,5′-trideoxycytidine-5′- phosphonic acid (ddC phosphonate)

Kofoed, T.; Ismail, A. E. A. A.; Pedersen, E. B.; Nielsen, C., Bull.Soc. Chim. Fr. 1997, 134: 59-65. 9-[3-(phosphono- methoxy)propyl]adenine(Adefovir)

Kim, C. U.; Luh, B. Y.; Misco, P. F.; Bronson, J. J.; Hitchcock, M. J.M.; Ghazzouli, I.; Martin, J. C., J. Med. Chem. 1990, 33: 1207-1213.

Some examples of antiviral phosphonates derived by substituting—CH₂—PO₃H₂ for the 5′-hydroxyl are:

Ganciclovir phosphonate

Huffman, J. H.; Sidwell, R. W.; Morrison, A. G.; Coombs, J.; Reist, E.J., Nucleoside Nucleotides, 1994, 13: 607-613. Acyclovir phosphonate

″ Ganciclovir cyclic phosphonate

Smee, D. F.; Reist, E. J., Antimicrob. Agents Chemother. 1996, 40: 1964-1966. 3′-thia-2′,3′- dideoxycytidine-5′- phosphonic acid

Kraus, J. L.; Nucleosides Nucleotides, 1993, 12: 157- 162.Other preferred antiviral nucleotide phosphonates contemplated for usein the practice of the invention are derived similarly from antiviralnucleosides including ddA, ddI, ddG, L-FMAU, DXG, DAPD, L-dA, L-dl,L-(d)T, L-dC, L-dG, FTC, penciclovir, and the like.

Additionally, antiviral phosphonates such as cidofovir, cycliccidofovir, adefovir, tenofovir, and the like, may be used as an R₃ groupin accordance with the present invention.

Certain compounds of the invention possess one or more chiral centers,e.g. in the sugar moieties, and may thus exist in optically activeforms. Likewise, when the compounds contain an alkenyl group or anunsaturated alkyl or acyl moiety there exists the possibility of cis-and trans-isomeric forms of the compounds. Additional asymmetric carbonatoms can be present in a substituent group such as an alkyl group. TheR- and S-isomers and mixtures thereof, including racemic mixtures aswell as mixtures of cis- and trans-isomers are contemplated by thisinvention. All such isomers as well as mixtures thereof are intended tobe included in the invention. If a particular stereoisomer is desired,it can be prepared by methods well known in the art by usingstereospecific reactions with starting materials that contain theasymmetric centers and are already resolved or, alternatively, bymethods that lead to mixtures of the stereoisomers and resolution byknown methods.

Many phosphonate compounds exist that can be derivatized according tothe invention to improve their pharmacologic activity, or to increasetheir oral absorption, such as, for example, the compounds disclosed inthe following patents, each of which are hereby incorporated byreference in their entirety: U.S. Pat. No. 3,468,935 (Etidronate), U.S.Pat. No. 4,327,039 (Pamidronate), U.S. Pat. No. 4,705,651 (Alendronate),U.S. Pat. No. 4,870,063 (Bisphosphonic acid derivatives), U.S. Pat. No.4,927,814 (Diphosphonates), U.S. Pat. No. 5,043,437 (Phosphonates ofazidodideoxynucleosides), U.S. Pat. No. 5,047,533 (Acyclic purinephosphonate nucleotide analogs), U.S. Pat. No. 5,142,051(N-Phosphonylmethoxyalkyl derivatives of pyrimidine and purine bases),U.S. Pat. No. 5,183,815 (Bone acting agents), U.S. Pat. No. 5,196,409(Bisphosphonates), U.S. Pat. No. 5,247,085 (Antiviral purine compounds),U.S. Pat. No. 5,300,671 (Gem-diphosphonic acids), U.S. Pat. No.5,300,687 (Trifluoromethylbenzylphosphonates), U.S. Pat. No. 5,312,954(Bis- and tetrakis-phosphonates), U.S. Pat. No. 5,395,826 (Guanidinealky1-1,1-bisphosphonic acid derivatives), U.S. Pat. No. 5,428,181(Bisphosphonate derivatives), U.S. Pat. No. 5,442,101(Methylenebisphosphonic acid derivatives), U.S. Pat. No. 5,532,226(Trifluoromethybenzylphosphonates), U.S. Pat. No. 5,656,745 (Nucleotideanalogs), U.S. Pat. No. 5,672,697 (Nuleoside-5′-methylene phosphonates),U.S. Pat. No. 5,717,095 (Nucleotide analogs), U.S. Pat. No. 5,760,013(Thymidylate analogs), U.S. Pat. No. 5,798,340 (Nucleotide analogs),U.S. Pat. No. 5,840,716 (Phosphonate nucleotide compounds), U.S. Pat.No. 5,856,314 (Thio-substituted, nitrogen-containing, heterocyclicphosphonate compounds), U.S. Pat. No. 5,885,973 (olpadronate), U.S. Pat.No. 5,886,179 (Nucleotide analogs), U.S. Pat. No. 5,877,166(Enantiomericaily pure 2-aminopurine phosphonate nucleotide analogs),U.S. Pat. No. 5,922,695 (Antiviral phosphonomethoxy nucleotide analogs),U.S. Pat. No. 5,922,696 (Ethylenic and allenic phosphonate derivativesof purines), U.S. Pat. No. 5,977,089 (Antiviral phosphonomethoxynucleotide analogs), U.S. Pat. No. 6,043,230 (Antiviral phosphonomethoxynucleotide analogs), U.S. Pat. No. 6,069,249 (Antiviral phosphonomethoxynucleotide analogs); Belgium Patent No. 672205 (Clodronate); EuropeanPatent No. 753523 (Amino-substituted bisphosphonic acids); EuropeanPatent Application 186405 (geminal diphosphonates); and the like.

Certain bisphosphonate compounds have the ability to inhibit squalenesynthase and to reduce serum cholesterol levels in mammals, includingman. Examples of these bisphosphonates are disclosed, for example, inU.S. Pat. Nos. 5,441,946 and 5,563,128 to Pauls et al. Phosphonatederivatives of lipophilic amines, both of which are hereby incorporatedby reference in their entirety. Analogs of these squalene synthaseinhibiting compounds according to the invention, and their use in thetreatment of lipid disorders in humans are within the scope of thepresent invention. Bisphosphonates of the invention may be used orallyor topically to prevent or treat periodontal disease as disclosed inU.S. Pat. No. 5,270,365, hereby incorporated by reference in itsentirety.

As used herein, the term “alkyl” refers to a monovalent straight orbranched chain or cyclic radical of from one to twenty-four carbonatoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

As used herein, “substituted alkyl” comprises alkyl groups furtherbearing one or more substituents selected from hydroxy, alkoxy (of alower alkyl group), mercapto (of a lower alkyl group), cycloalkyl,substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl,substituted aryl, heteroaryl, substituted heteroaryl, aryloxy,substituted aryloxy, halogen, trifluoromethyl, cyano, nitro, nitrone,amino, amino, —C(O)H, acyl, oxyacyl, carboxyl, carbamate, sulfonyl,sulfonamide, sulfuryl, and the like.

As used herein, “alkenyl” refers to straight or branched chainhydrocarbyl groups having one or more carbon-carbon double bonds, andhaving in the range of about 2 up to 24 carbon atoms, and “substitutedalkenyl” refers to alkenyl groups further bearing one or moresubstituents as set forth above.

As used herein, “aryl” refers to aromatic groups having in the range of6 up to 14 carbon atoms and “substituted aryl” refers to aryl groupsfurther bearing one or more substituents as set forth above.

As used herein, “heteroaryl” refers to aromatic groups containing one ormore heteroatoms (e.g., N, O, S, or the like) as part of the ringstructure, and having in the range of 3 up to 14 carbon atoms and“substituted heteroaryl” refers to heteroaryl groups further bearing oneor more substituents as set forth above.

As used herein, the term “bond” or “valence bond” refers to a linkagebetween atoms consisting of an electron pair.

As used herein, the term “pharmaceutically acceptable salts” refers toboth acid and base addition salts.

As used herein, the term “prodrug” refers to derivatives ofpharmaceutically active compounds that have chemically or metabolicallycleavable groups and become the pharmaceutically active compound bysolvolysis or under in vivo physiological conditions.

Phosphonate analogs, comprising therapeutically effective phosphonates(or phosphonate derivatives of therapeutically effective compounds)covalently linked by a hydroxyl group to a 1-O-alkyglycerol,3-O-alkylglycerol, 1-S-alkylthioglycerol, or alkoxy-alkanol, may beabsorbed more efficiently in the gastrointestinal tract than are theparent compounds. An orally administered dose of the analog is taken upintact from the gastrointestinal tract of a mammal and the active drugis released in vivo by the action of endogenous enzymes. Phosphonateanalogs of the invention may also have a higher degree of bioactivitythan the corresponding underivatized compounds.

The compounds of the present invention are an improvement overalkylglycerol phosphate prodrugs described in the prior art because thephosphonate-containing moiety is linked directly to the alkyl-glycerolor the alkoxy-alkanol moiety and because the presence of the phosphonatebond prevents enzymatic conversion to the free drug. Other linkersbetween these groups can be present in the improved analogs. Forexample, bifunctional linkers having the formula —O—(CH₂)_(n)—C(O)O—₉wherein n is 1 to 24, can connect the phosphonate to the hydroxyl groupof the alkoxy-alkanol or alkylglycerol moiety.

The foregoing allows the phosphonate of the invention to achieve ahigher degree of oral absorption. Furthermore, cellular enzymes, but notplasma or digestive tract enzymes, will convert the conjugate to a freephosphonate. A further advantage of the alkoxy-alkanol phosphonates isthat the tendency of co-administered food to reduce or abolishphosphonate absorption is greatly reduced or eliminated, resulting inhigher plasma levels and better compliance by patients.

Compounds of the invention can be administered orally in the form oftablets, capsules, solutions, emulsions or suspensions, inhaled liquidor solid particles, microencapsulated particles, as a spray, through theskin by an appliance such as a transdermal patch, or rectally, forexample, in the form of suppositories. The lipophilic prodrugderivatives of the invention are particularly well suited fortransdermal absorption administration and delivery systems and may alsobe used in toothpaste. Administration can also take place parenterallyin the form of injectable solutions.

The compositions may be prepared in conventional forms, for example,capsules, tablets, aerosols, solutions, suspensions, or together withcarriers for topical applications. Pharmaceutical formulationscontaining compounds of this invention can be prepared by conventionaltechniques, e.g., as described in Remington's Pharmaceutical Sciences,1985.

The pharmaceutical carrier or diluent employed may be a conventionalsolid or liquid carrier. Examples of solid carriers are lactose,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate,stearic acid, or lower alkyl ethers of cellulose. Examples of liquidcarriers are syrup, peanut oil, olive oil, phospholipids, fatty acids,fatty acid amines, polyoxyethylene or water. The carrier or diluent mayinclude any sustained release material known in the art, such asglyceryl monostearate or distearate, alone or mixed with a wax.

If a solid carrier is used for oral administration, the preparation maybe tabletted or placed in a hard gelatin capsule in powder or pelletform. The amount of solid carrier will vary widely, but will usually befrom about 25 mg to about 1 gm. If a liquid carrier is used, thepreparation may be in the form of a syrup, emulsion, soft gelatincapsule, or sterile injectable liquid such as an aqueous or non-aqueousliquid suspension or solution.

Tablets are prepared by mixing the active ingredient (that is, one ormore compounds of the invention), with pharmaceutically inert, inorganicor organic carrier, diluents, and/or excipients. Examples of suchexcipients which can be used for tablets are lactose, maize starch orderivatives thereof, talc, stearic acid or salts thereof. Examples ofsuitable excipients for gelatin capsules are vegetable oils, waxes,fats, semisolid, and liquid polyols. The bisphosphonate prodrugs canalso be made in microencapsulated form.

For nasal administration, the preparation may contain a compound of theinvention dissolved or suspended in a liquid carrier, in particular, anaqueous carrier, for aerosol application. The carrier may containsolubilizing agents such as propylene glycol, surfactants, absorptionenhancers such as lecithin or cyclodextrin, or preservatives.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or non-aqueousliquids, dispersions, suspensions or emulsions as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use.

Suitable excipients for the preparation of solutions and syrups arewater, polyols, sucrose, invert sugar, glucose, and the like. Suitableexcipients for the preparation of injectable solutions are water,alcohols, polyols, glycerol, vegetable oils, and the like.

The pharmaceutical products can additionally contain any of a variety ofadded components, such as, for example, preservatives, solubilizers,stabilizers, wetting agents, emulsifiers, sweeteners, colorants,flavorings, buffers, coating agents, antioxidants, diluents, and thelike.

Optionally, the pharmaceutical compositions of the invention maycomprise a compound according to the general formula combined with oneor more compounds exhibiting a different activity, for example, anantibiotic or other pharmacologically active material. Such combinationsare within the scope of the invention.

This invention provides methods of treating mammalian disorders relatedto bone metabolism, viral infections, inappropriate cell proliferation,and the like. The methods particularly comprise administering to a humanor other mammal in need thereof a therapeutically effective amount ofthe prodrugs of this invention. Indications appropriate to suchtreatment include senile, post-menopausal or steroid-inducedosteoporosis, Paget's disease, metastatic bone cancers,hyperparathyroidism, rheumatoid arthritis, algodystrophy,stemo-costoclavicular hyperostosis, Gaucher's disease, Engleman'sdisease, certain non-skeletal disorders and periodontal disease, humanimmunodeficiency virus (HIV), influenza, herpes simplex virus (HSV),human herpes virus 6, cytomegalovirus (CMV), hepatitis B virus,Epstein-Barr virus (EBV), varicella zoster virus, lymphomas,hematological disorders such as leukemia, and the like.

In accordance with one aspect of the invention, there are providedmethods of preventing or treating bone loss in mammals, especiallyhumans, which method comprises” administering to the human or mammal atherapeutically effective amount of the compounds of this invention. Thebone resorption inhibiting bisphosphonate prodrugs of the invention areuseful therapeutically to oppose osteoclast-mediated bone resorption orbone loss in conditions wherein the bisphosphonate from which theprodrug is prepared has been found efficacious. Indications appropriateto such treatment include osteoporosis, particularly in postmenopausalwomen, the osteoporosis that accompanies long-term glucocortcoidtherapy, and Paget's disease of bone. The bisphosphonate compoundclodronate (Ostac, Boehringer-Mannheim, Mannheim, Germany) has also beenfound to reduce osseous as well as visceral metastases in breast cancerpatients at high risk for distant metastases (Diel, I. J. et al. (1998)New Engl. J. Med. 339(60 357-363). Efficacy of the bisphosphonateprodrugs of the invention can be evaluated according to the same methodsas for the parent compound. These comprise comparative measurement ofbone mineral density of the lumber spine, femoral neck, trochanter,forearm and total body, together with measurements of vertebralfractures, spinal deformities and height in osteoporosis, bone scans orradiographic identification of bone lesions in metastatic disease, andthe like.

In accordance with another aspect of the invention, there are providedmethods for increasing bone mass and strength in mammals, especiallyhumans, by administering bone anabolism-promoting compounds of theinvention which inhibit osteoblast and osteocyte apoptosis, leading togreater net rates of bone formation, while not substantially alteringosteoclast functions (Plotkin et al., J Clin Invest 104:1363-1374 (1999)and Van Beek et al., J Bone Min Res 11:1492 (1996)).

In accordance with yet another aspect of the invention, there areprovided methods for treating disorders caused by viral infections.Indications appropriate to such treatment include susceptible virusessuch as human immunodeficiency virus (HIV), influenza, herpes simplexvirus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis Band C virus, Epstein-Barr virus (EBV), varicella zoster virus, anddiseases caused by orthopox viruses (e.g., variola major and minor,vaccinia, smallpox, cowpox, camelpox, monkeypox, and the like), ebolavirus, papilloma virus, and the like.

In accordance with still another aspect of the invention, there areprovided methods for treating disorders caused by inappropriate cellproliferation, e.g. cancers, such as melanoma, lung cancers, pancreaticcancer, stomach, colon and rectal cancers, prostate and breast cancer,the leukemias and lymphomas, and the like. Anti-cancer compounds whichcan be converted to their nucleotide phosphonates for use as compoundsof this invention include, but are not limited to, cytarabine (ara-C),fluorouridine, fluorodeoxyuridine (floxuridine), gemcitibine,cladribine, fludarabine, pentostatin (2′-deoxycoformycin),6-mercaptopurine and 6-thioguanine and substituted or unsubstitutedara-adenosine (ara-A), ara-guanosine (ara-G), and ara-uridine (ara-U).Anticancer compounds of the invention may be used alone or incombination with other antimetabobtes or with other classes ofanticancer drugs such as alkaloids, topoisomerase inhibitors, alkylatingagents, antifumor antibiotics, and the like.

The prodrugs of the invention can be administered orally, parenterally,topically, rectally, and through other routes, with appropriate dosageunits, as desired.

As used herein, the term “parenteral” refers to subcutaneous,intravenous, intra-arterial, intramuscular or intravitreal injection, orinfusion techniques.

The term “topically” encompasses administration rectally and byinhalation spray, as well as the more common routes of the skin andmucous membranes of the mouth and nose and in toothpaste.

The term “effective amount” as applied to the phosphonate prodrugs ofthe invention is an amount that will prevent or reverse the disordersnoted above. Particularly with respect to disorders associated with bonemetabolism, an effective amount is an amount that will prevent,attenuate, or reverse abnormal or excessive bone resorption or the boneresorption that occurs in the aged, particularly post-menopausal femalesor prevent or oppose bone metastasis and visceral metastasis in breastcancer.

With respect to disorders associated with viral infections orinappropriate cell proliferation, e.g., cancer, the “effective amount”is determined with reference to the recommended dosages of the antiviralor anticancer parent compound. The selected dosage will vary dependingon the activity of the selected compound, the route of administration,the severity of the condition being treated, and the condition and priormedical history of the patient being treated. However, it is within theskill of the art to start doses of the compound(s) at levels lower thanrequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. If desired,the effective daily dose may be divided into multiple doses for purposesof administration, for example, two to four doses per day. It will beunderstood, however, that the specific dose level for any particularpatient will depend on a variety of factors, including the body weight,general health, diet, time, and route of administration and combinationwith other drugs, and the severity of the disease being treated.

Generally, the compounds of the present invention are dispensed in unitdosage form comprising 1% to 100% of active ingredient. The range oftherapeutic dosage is from about 0.01 to about 1,000 mg/kg/day with fromabout 0.10 mg/kg/day to 100 mg/kg/day being preferred, when administeredto patients, e.g., humans, as a drug. Actual dosage levels of activeingredients in the pharmaceutical compositions of this invention may bevaried so as to administer an amount of the active compound(s) that iseffective to achieve the desired therapeutic response for a particularpatient.

A number of animal experiments have shown the efficacy ofbisphosphonates in preventing bone loss under experimental conditionsdesigned to mimic relevant clinical disorders. Based on these studies,several small animal model systems are available for evaluating theeffects of bisphosphonates. These tests are also useful for measuringthe comparative efficacy of the bisphosphonate prodrugs of theinvention. The evaluation of bisphosphonate therapy typically requiresthe determination of femoral ash weight and bone mass, measured, forexample as trabecular bone volume, between groups of treated anduntreated animals. Thompson, D. et al. (1990) J. Bone and Mineral Res.5(3):279-286, discloses use of such methods for evaluating theinhibition of bone loss in immobilized rats that were treated withaminohydroxybutane bisphosphonate. Yamamoto, M. et al. (1993) CalcifTissue Int 53:278-282 induced hyperthyroidism in rats to produce bonechanges similar to those in hyperthyroid humans, and comparedbisphosphonate-treated and untreated groups biochemically, based onosteocalcin measurement, and by histomorphometric analysis, includingdifferences in cancellous bone volume, and histological comparison ofosteoid, osteoclast and osteoblast surfaces in bone sections. Seedor, J.G. et al. (1991) J. Bone and Mineral Res. 6(4):339-346 describes studiesof the effect of alendronate in opposing bone loss in overactomized ratsby femoral ash weight and histomorphometric analysis of tibialtrabecular volume. The Schenk assay, comprising histological examinationof the epiphyses of growing rats, can also be used as a screening assay.An exemplary screening test for evaluating the bone resorption opposingeffects of the compounds of the invention in laboratory rats madeosteopenic by various strategies is provided in Example 14.

Compounds of the invention can be prepared in a variety of ways, asgenerally depicted in Schemes I-VI. The general phosphonateesterification methods described below are provided for illustrativepurposes only and are not to be construed as limiting this invention inany manner. Indeed, several methods have been developed for directcondensation of phosphonic acids with alcohols (see, for example, R. C.Larock, Comprehensive Organic Transformations, VCH, New York, 1989, p.966 and references cited therein). Isolation and purification of thecompounds and intermediates described in the examples can be effected,if desired, by any suitable separation or purification procedure suchas, for example, filtration, extraction, crystallization, flash columnchromatography, thin-layer chromatography, distillation or a combinationof these procedures. Specific illustrations of suitable separation andisolation procedures are in the examples below. Other equivalentseparation and isolation procedures can of course, also be used.

Scheme I outlines a synthesis of bisphosphonate prodrugs that contain aprimary amino group, such as pamidronate or alendronate. Example 1provides conditions for a synthesis of1-O-hexadecyloxypropyl-alendronate (HDP-alendronate) or1-O-hexadecyloxypropyl-pamidronate (HDP-pamidronate). In this process, amixture oT dimethyl 4-phthalimidobutanoyl phosphonate (1b, prepared asdescribed in U.S. Pat. No. 5,039,819)) and hexadecyloxypropyl methylphosphite (2) in pyridine solution is treated with triethylamine toyield bisphosphonate tetraester 3b which is purified by silica gelchromatography. Intermediate 2 is obtained by transesterification ofdiphenyl phosphite as described in Kers, A., Kers, I., Stawinski, J.,Sobkowski, M., Kraszewski, A. Synthesis, April 1995, 427-430. Thus,diphenyl phosphite in pyridine solution is first treated withhexadecyloxypropan-1-ol, then with methanol to provide compound 2.

An important aspect of the process is that other long chain alcohols maybe used in place of hexadecyloxypropan-1-ol to generate the variouscompounds of this invention. Treatment of intermediate 3b withbromotrimethylsilane in acetonitnle cleaves the methyl estersselectively to yield monoester 4b. Treatment of 4b with hydrazine in amixed solvent system (20% melhanol/80% 1,4-dioxane) results in removalof the phthalimido protecting group as shown. The desired alendronateprodrug is collected by filtration and converted to the triammonium saltby treatment with methanolic ammonia.

Scheme II illustrates a synthesis of analogs of bisphosphonates lackinga primary amino group, hi this case the process steps are similar tothose of Scheme 1 except that protection with a phthalimido group andsubsequent deprotection by hydrazinolysis are unnecessary.

Bisphosphonates having 1-amino groups, such as amino-olpadronate, maybeconverted to analogs according to the invention prodrugs using aslightly modified process shown in Scheme III.

Treatment of a mixture of compound 2 and 3-(dimethylamino)propionitrilewith dry HCl followed by addition of dimethyl phosphite affordstetraester 3 which, after demethylation with bromotrimethylsilane,yields hexadecyloxypropyl-amino-olpadronate.

Scheme IV illustrates synthesis of a bisphosphonate analog where thelipid group is attached to a primary amino group of the parent compoundrather than as a phosphonate ester.

Scheme V illustrates a general synthesis of alkylglycerol oralkylpropanediol analogs of cidofovir, cyclic cidofovir, and otherphosphonates. Treatment of 2,3-isopropylidene glycerol, 1, with NaH indimethylformamide followed by reaction with an alkyl methanesulfonateyields the alkyl ether, 2. Removal of the isopropylidene group bytreatment with acetic acid followed by reaction with trityl chloride inpyridine yields the intermediate 3. Alkylation of intermediate 3 with analkyl halide results in compound 4. Removal of the trityl group with 80%aqueous acetic acid affords the O,O-dialkyl glycerol, 5. Bromination ofcompound 5 followed by reaction with the sodium salt of cyclic cidofoviror other phosphonate-containing nucleotide yields the desiredphosphonate adduct, 7. Ring-opening of the cyclic adduct is accomplishedby reaction with aqueous sodium hydroxide. The preferred propanediolspecies may be synthesized by substituting 1-O-alkylpropane-3-ol forcompound 5 in Scheme V. The tenofovir and adefovir analogs may besynthesized by substituting these nucleotide phosphonates for cCDV inreaction (f) of Scheme V. Similarly, other nucleotide phosphonates ofthe invention may be formed in this manner.

Scheme VI illustrates a general method for the synthesis of nucleotidephosphonates of the invention using 1-O-hexadecyloxypropyl-adefovir asthe example. The nucleotide phosphonate (5 mmol) is suspended in drypyridine and an alkoxyalkanol or alkylglycerol derivative (6 mmol) and1,3-dicyclohexylcarbodiimde (DCC, 10 mmol) are added. The mixture isheated to reflux and stirred vigorously until the condensation reactionis complete as monitored by thin-layer chromatography. The mixture isthen cooled and filtered. The filtrate is concentrated under reducedpressure and the residue s adsorbed on silica gel and purified by flashcolumn chromatography (elution with approx. 9:1dichloromethane/methanol) to yield the corresponding phosphonatemonoester.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

Example 1 Synthesis of L-O-Hexadecylpropanediol-3-Alendronate A.Hexadecyloxypropyl Methyl Phosphite (b)

Hexadecyloxypropyl methyl phosphite was prepared using the methoddescribed in: Kers, A., Kers, I., Stawinski, J., Sobkowski, M.,Kraszewski, A. Synthesis April 1995, 427-430. To a solution ofdiphenylphosphite (14 g, 60 mmol) in pyridine (50 mL) maintained at 0°C. was slowly added to a solution of hexadecyloxypropan-1-ol (6.0 g, 20mmol) in pyridine (25 mL). The mixture was stirred one hour beforeanhydrous methanol (10 mL) was added. After stirring an additional hour,the solvent was evaporated the residue was adsorbed on silica gel andchromatographed, using gradient elution (hexanes to 20% ethylacetate/80% hexanes), to afford pure compound 2 as a waxy, low-meltingsolid (4.5 g, 60% yield). ¹H NMR (CDCl₃) δ 6.79 (d, 1H, J=696 Hz), 4.19(q, 2H), 3.78 (d, 3H), 3.51 (t, 3H), 3.40 (t, 2H), 1.95 (pent, 2H), 1.25(broad s, 28H), 0.88 (t, 3H).

B. Hexadecyloxypropyl trimethyl 4-phthalimidobutanoyl phosphonate (3b)

To a mixture of dimethyl 4-phthalimidobutanoyl phosphonate (1b, 3.0 g,7.9 mmol, prepared as described in U.S. Pat. No. 5,039,819) andhexadecyloxypropyl methyl phosphite (2, 2.9 g, 9 mmol) in pyridine (50mL) was added triethylamine (0.2 g, 2 mmol). The mixture was stirred 5hours at room temperature, then the solvent was removed in vacuo. Theresidue was adsorbed on silica gel and chromatographed (ethyl acetate)to give compound 3b (3.5 g, 63%) as a viscous oil. ¹H NMR (CDCl₃) δ 7.84(d, 2H), 7.72 (d, 2H), 4.45 (m, 1H), 4.27 (m, 4H), 4.15 (q, 2H), 3.68(s, 3H), 3.84 (s, 3H), 3.71 (t, 2H), 3.51 (m, 2H), 3.38 (t, 2H), 2.04(m, 2H), 1.94 (pent., 2H), 1.54 (m, 2H), 1.25 (broad s, 28H), 0.88 (t,3H). ³¹P NMR (22.54 (doublet), 21.22 (quartet)).

C. Hexadecyloxypropyl 4-phthalimidobutanoyl phosphonate (4b)

Compound 3b from above (3.0 g, 4.3 mmol) was dissolved in dryacetonitrile (50 mL) and cooled to 0° C. A solution ofbromotrimethylsilane (3.9 g, 25.5 mmol) in acetonitrile (25 mL) wasadded slowly then the solution was stirred an additional 2 hours. Themixture was then poured slowly into crushed ice. The precipitate thatformed was collected by vacuum filtration and dried in vacuo to give 1.2g of 4b (42% yield). ¹H NMR (DMSO-d6) 7.86 (m, 4H), 3.99 (q, 2H),3.66-3.55 (m, 1H), 3.54 (m, 2H), 3.35 (t, 2H), 3.27 (t, 2H), 1.89-1.80(m), 1.72 (pent., 2H), 1.53-1.40 (m, 2H), 1.22 (broad s, 28H), 0.85 (t,3H). ³¹P NMR (21.51 (doublet), 19.50 (doublet)).

D. 1-O-Hexadececylpropanediol-3-alendronate (5b)

Compound 4b (300 mg, 0.45 mmol) was dissolved in a mixture of1,4-dioxane (20 mL) and methanol (5 mL). Anhydrous hydrazine was thenadded and the mixture was stirred at room temperature for 4 hours. Theprecipitate that separated was collected by vacuum filtration and rinsedwith 1,4-dioxane. The solid was then suspended in ethanol and methanolicammonia (3 mL) was added. After stirring for 10 minute's the resultingsolid was collected by filtration, rinsed with ethanol and dried undervacuum to yield 220 mg HDP-alendronate (5b) as the triammonium salt.Analysis by FT-IR indicated removal of the phthalimido protecting group.Electrospray MS m/e 532 (MH+), 530 (MH).

Example 2 Synthesis of L-O-Bexadecyipropanediol-3-Pamidronate (5a)

1-O-hexadecylpropanediol-3-pamidronate is prepared in an analogousmanner (according to Scheme 1) except that 3-phthalimidopropanoic acidis used to prepare dimethyl 3-phthalimidopropanoyl phosphonate (1a).Compound 1a is condensed with 2 to yield the trimethyl bisphosphonate3a. Deprotection as in Steps C and D above yields HDP-pamidronate asshown.

Example 3 Synthesis of L-O-Octadecyl-2-O-Methyl Sn-Glycero-3-Alendronate

Prodrugs with lipophilic groups other than hexadeclyoxypropyl areprepared by substituting various long-chain alcohols forhexadecyloxypropan-1-ol in Step A of Example 1. For example, reaction of1-O-Octadecyl-2-O-methyl-sn-glycerol with diphenylphosphite in pyridinefollowed by treatment with methanol gives1-O-octadecyl-2-O-methyl-sn-glyceryl methyl phosphite. Condensation ofthis dialkylphosphite with phosphonate 1b, followed by deprotectionsteps C and D gives 1-O-Octadecyl-2-O-methyl-sn-glycero-3-alendronate.Scheme 2 illustrates a synthesis of other bisphosphonate conjugateswhich do not have a primary amino group in the side chain. In this caseprotection with a phthalimido group and deprotection by hydrazinolysisare unnecessary.

Example 4 Synthesis of HDP-Amino-Olpadronate

Scheme 3 illustrates the synthesis of 1-amino bisphosphonate conjugates.Using compound 2 from Example 1, 3-(dimethylamino)propionitrile, andprocedures described in: Orlovskii, V. V.; Vovsi, B. A. J. Gen Chem.USSR (Engl. Transl.) 1976, 46: 294-296, the bisphosphonate trimethylester 3 is prepared. Demethylation with bromotrimethylsilane asdescribed in step C of Example 1 provides HDP-amino-olpadronate.

Example 5 Synthesis of L-O-Hexadecylpropanediol-3-Succinyl-Alendronate

Scheme 4 illustrates the synthesis of a bisphosphonate conjugate whereinthe lipid group is attached to a primary amino group of the parentcompound. Tetramethyl-(4-phthalimido-1-hydroburylidene)bisphosphonate(2.0 g, 4.4 mmol) was dissolved in 0.2M methanolic hydrazine (100 mL),and the solution stirred at room temperature for 3 days. The mixture wasconcentrated to half its volume when a solid started separating. Thesolid was filtered off and the filtrate concentrated to dryness. ProtonNMR showed this compound to betetramethyl-(4-amino-1-hydroxybutylidene)bisphosphonate. This was driedover phosphorus pentoxide at 50° C. overnight. To a suspension of 1.2 gof the compound in a mixture of pyridine (25 mL) andN,N-dimethylformamide (25 mL) was added 3-succinyl-1-hexdeclyoxypropane(1.76 g, 4.4 mmol). Dicyclohexyl carbodiimide (2.52 g, 12.21 mmol) wasadded and the mixture stirred at room temperature for two days. Themixture was filtered; the filtrate was absorbed on silica gel and flashchromatographed with an increasing gradient of methanol indichloromethane (0%-20%) to yield succinylated compound. This wasdeblocked with trimethylsilyl bromide in acetronitrile to yield thetitle compound which was purified by crystallization from methanol.

Example 6 Synthesis of Adefovir Hexadecyjoxypropyl and1-O-Octadecyi-Sn-Glyceryl Esters

To a mixture of adefovir (1.36 g, 5 mmol) and 3-hexadecyloxy-1-propanol(1.8 g, 6 mmol) in dry pyridine was added DCC (2.06 g, 10 mmol). Themixture was heated to reflux and stirred 18 h then cooled and filtered.The filtrate was concentrated under reduced pressure and the residue wasapplied to a short column of silica gel. Elution of the column with 9:1dichloromethane/methanol yielded hexadecyloxypropyl-adefovir (HDP-ADV)as a white powder.

To a mixture of adefovir (1.36 g, 5 mmol) and 1-O-octadecyl-sn-glycerol(2.08 g, 6 mmol) in dry pyridine (30 mL) was added DCC (2.06 g, 10mmol). The mixture was heated to reflux and stirred overnight thencooled and filtered. The filtrate was concentrated under reducedpressure and the residue was applied to a column of silica gel. Elutionof the column with a 9:1 dichloromethane/methanol yielded1-O-octadecyl-sn-glyceryl-3-adefovir.

Example 7 Synthesis of AZT-Phosphonate Hexadecyloxypropyl Ester

The phosphonate analog of AZT(3′-Azido-S′—S′-dideoxythymidine-S′-phosphonic acid) was synthesizedusing the published procedure: Hakimelahi, G. H.; Moosavi-Movahedi, A.A.; Sadeghi, M. M.; Tsay, S-C; Hwu, J. R. Journal of MedicinalChemistry, 1995 38, 4648-4659.

The AZT phosphonate (1.65 g, 5 mmol) was suspended in dry pyridine (30mL), then 3-hexadecyloxy-1-propanol (1.8 g, 6 mmol) and DCC (2.06 g, 10mmol) were added and the mixture was heated to reflux and stirred for 6h, then cooled and filtered. The filtrate was concentrated under reducedpressure and the residue was applied to a column of silica gel. Elutionof the column with a 9:1 dichloromethane/methanol yielded3′-azido-3′-5′-dideoxythymidine-5′-phosphonic acid, hexadecyloxypropylester.

Example 8 Synthesis of the Hexadecyloxypropyl, Octadecyloxypropyl,Octadecyloxyethyl and Hexadecyl Esters of Cyclic Cidofovir

To a stirred suspension of cidofovir (1.0 g, 3.17 mmol) in N,N-DMF (25mL) was added N,N-dicyclohexyl-4-morpholine carboxamidine (DCMC, 1.0 g,3.5 mmol). The mixture was stirred overnight to dissolve the cidofovir.This clear solution was then charged to an addition funnel and slowlyadded (30 min.) to a stirred, hot pyridine solution (25 mL, 60° C.) of1,3-dicyclohexyl carbodiimide (1.64 g, 7.9 mmol). This reaction mixturewas stirred at 100° C. for 16 h then cooled to room temperature, and thesolvent was removed under reduced pressure. The residue was adsorbed onsilica gel and purified by flash column chromatography using gradientelution (CH₂Cl₂+MeOH). The UV active product was finally eluted with5:5:1 CH₂Cl₂/MeOH/H₂O Evaporation of the solvent gave 860 mg of a whitesolid. The ¹H and ³¹P NMR spectrum showed this to be the DCMC salt ofcyclic cidofovir (yield=44%).

To a solution of cyclic cidofovir (DCMC salt) (0.5 g, 0.8 mmol) in dryDMF (35 mL) was added 1-bromo-3-hexadecyloxypropane (1.45 g, 4 mmol) andthe mixture was stirred and heated at 80° C. for 6 h. The solution wasthen concentrated in vacuo and the residue adsorbed on silica gel andpurified by flash column chromatography using gradient elution(CH₂CI₂+EtOH). The alkylated product was eluted with 90:10 CH₂CI₂/EtOH.The fractions containing pure product were evaporated to yield 260 mgHDP-cyclic cidofovir (55% yield).

To a solution of cyclic cidofovir (DCMC salt) (1.0 g, 3.7 mmol) in dryDMF (35 mL) was added 1-bromo-3-octadecyloxypropane (2.82 g, 7.2 mmol)and the mixture was stirred and heated at 85° C. for 5 h. The solutionwas then concentrated in vacuo and the residue adsorbed on silica geland purified by flash column chromatography using gradient elution(CH2CI2+MeOH). The alkylated product was eluted with 9:1 CH₂CI₂/MeOH.The fractions containing pure product were evaporated to yield 450 mgODP-cyclic cidofovir.

To a solution of cCDV (DCMC salt) (1.0 g, 3.7 mmol) in dry DMF (35 mL)was added 1-bromo-3-octadecyloxyethane (3.0 g, 7.9 mmol) and the mixturewas stirred and heated at 80° C. for 4 h. The solution was thenconcentrated in vacuo and the residue adsorbed on silica gel andpurified by flash column chromatography using gradient elution(CH₂Cl₂+MeOH). The alkylated product was eluted with 9:1 CH₂CI₂/MeOH.The fractions containing pure product were evaporated to yield 320 mgoctadecyloxyethyl-cCDV.

To a solution of cyclic cidofovir (DCMC salt) (0.5 g, 0.8 mmol) in dryDMF (35 mL) was added 1-bromo-hexadecane (1.2 g, 4 mmol) and the mixturewas stirred and heated at 80° C. for 6 h. The solution was thenconcentrated in vacuo and the residue adsorbed on silica gel andpurified by flash column chromatography using gradient elution(CH₂CI₂+MeOH). The alkylated product was eluted with 9:1 CH₂CI₂/MeOH.The fractions containing pure product were evaporated to yield 160 mghexadecyl-cCDV.

Example 9 Synthesis of the Hexadecyloxypropyl, Octadecyloxypropyl,Octadecyloxyethyl and Hexadecyl Esters of Cidofovir

Hexadecyloxypropyl-cyclic CDV from above was dissolved in 0.5 M NaOH andstirred at room temp for 1.5 h. 50% aqueous acetic was then addeddropwise to adjust the pH to about 9. The precipitated HDP-CDV wasisolated by filtration, rinsed with water and dried, then recrystallized(3:1 p-dioxane/water) to give HDP-CDV.

Similarly, the octadecyloxypropyl-, octadecyloxyethyl- andhexadecyl-cCDV esters were hydrolyzed using 0.5 M NaOH and purified togive the corresponding cidofovir diesters.

Example 10 Synthesis of Cyclic-Ganciclovir PhosphonateHexadecyloxypropyl Ester

The cyclic phosphonate analog of ganciclovir was prepared using thepublished procedure: (Reist, E. J.; Sturm, P. A.; Pong, R. Y.; Tanga, M.J. and Sidwell, R. W. Synthesis of acyclonucleoside phosphonates forevaluation as antiviral agents, p. 17-34. In J, C. Martin (ed.),Nucleotide Analogues as Antiviral Agents, American Chemical Society,Washington, D.C). After conversion to the DCMC salt in DMF the cGCVphosphonate was treated with 1-bromo-3-hexadecyloxypropane and themixture was heated to 80° C. for 6 hours. Isolation of the alkylatedproduct by flash chromatography yielded HDP-cyclic-GCV phosphonate.

Example 11 Synthesis of Ganciclovir Pbosphonate Hexadecyloxypropyl Ester

HDP-cyclic GCV phosphonate from above was dissolved in 0.5 M NaOH andstirred at room temperature to convert it to the acyclic diester. Thesolution was neutralized with 50% aq acetic acid to precpitate theproduct which was recrystallized in 3:1 p-dioxane/water.

Example 12 1-O-Hexadecyloxypropane Alendronate InhibitsDexametbasone-Induced Apoptosis of MLO-Y4 Osteocytic Cells

MLO-Y4 osteocytic cells were pretreated with the indicated concentrationof 1-O-hexadecyloxypropane alendronate (HDP-alendronate) for 1 hour, andsubsequently the cells were incubated for 6 hours with and withoutdexamethasone (10⁻⁴ M final concentration). The percentage of dead cellswas determined by trypan blue update (Plotkin et al., J Clin Invest104:1363-1374, 1999). Results are presented in FIG. 1. Bars representthe mean±SD of 3 independent measurements. Data were analyzed by 1-wayANOVA (Student-Keuls-Newman test). *p<0.05. HDP-alendronate inhibitsdexamethasone-induced apoptosis at 10⁻⁸ to 10⁻⁵ M.

Example 13 1-O-Hexadecyloxypropane Alendronate InhibitsDexamethasone-Induced Apoptosis in Calvarial Cells

Calvarial cells were obtained from neonatal C57BL/6J mice and passagedin tissue culture. The cells were pretreated with the indicatedconcentration of HDP-alendronate for 1 hour, and subsequently the cellswere incubated for 6 hours with and without 10⁻⁴ dexamethasone. Thepercentage of dead cells was determined by trypan blue uptake (Plotkin,L. et al., J Clin Invest 104:1363-1374, 1999). Results are presented inFIG. 2. Bars represent the mean±SD of 3 independent measurements. Datawere analyzed by 1-way ANOVA (Student-Keuls-Newman test). *p<0.05.Pretreatment of cells with HDP-alendronate at 10⁻⁸ or greater abolishedthe dexametEasone-induced increase in % dead cells (p=<0.05). Cellsexposed to 0.05 μM DEVD (a peptide inhibitor of apoptosis) followed bydexamethasone did not exhibit an increase in % dead cells demonstratingthat DEVD blocks dexamethasone-induced apoptosis.

Example 14 Inhibition of Bone Resorption in Ovariectomized Rats byL-O-Hexadecylpropane Alcndronate

Members of groups of (250 gm-280 gm) female Sprague-Dawley rats thathave undergone bilateral ovariectomy are treated either with4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid, disodium salt or1-O-hexadecylpropanediol-3-alendronate injected subcutaneously ingraduated doses of from 0 mg/kg/day to 8 mg/kg/day, for a period of fourto twelve weeks. At twelve weeks the rats, including members of acontrol group, are sacrificed and the femora of each animal is ashed.Alternatively, the method of administration may be oral. The ash weightof the femora for each individual is determined, the values for eachgroup compared as an indicator of bone mass to determine relativeinhibition of bone loss among the treatment protocols.1-O-hexadecylpropane alendronate-treated animals exhibit less bone massloss than the ovariectomized controls.

Example 15 Inhibition of Bone Resorption in Humans with Osteoporosis byL-O-Octadecyloxypropyl-Alendronate

Two groups of postmenopausal women are treated with placebo or with1-O-octadecyloxypropyl-alendronate at an oral dose of from 0.1 mg/kg/dayto 100 mg/kg/day for a period of from two to three years. Members of thetreatment groups are continually monitored over the course of treatmentfor bone mineral density, incidence of vertebral fractures, progressionof vertebral deformities by radiographic examination and height loss.Comparisons of measurements are made among the various treatment groupsto determine the effectiveness of the forms of alendronate therapy amongthe treatment group. The group treated with 1-O-octadecyloxypropylalendronate will have fewer fractures and a lesser rate of reduction inbone density than the placebo group.

Example 16 Stimulation of Bone Formation in Humans with Steroid-InducedOsteoporosis by 1-O-Octadecyloxypropyl-Amino-Olpadronate

Groups of patients with steroid-induced osteoporosis are treated with1-O-octadecyloxypropyl-amino-olpadronate or placebo at an oral dose offrom 0.1 mg/kg/day to 100 mg/kg/day for a period of from one month toone year. Members of the treatment groups are continually monitored overthe course of treatment for bone mineral density, incidence of vertebralfractures, progression of vertebral deformities by radiographicexamination and height loss. Comparisons of measurements are made amongthe various treatment groups to determine the effectiveness of1-O-octadecyloxypropyl-amino-olpadronate therapy among the treatmentgroup. Compared with placebo treatment, bone density is increased andfractures are decreased in1-O-octadecyloxypropyl-amino-olpadronate-treated patients.

Example 17 Antiviral Activity and Selectivity of Phosphonate NucleotideAnalogs Against Human Cytomegalovirns (HCMV)

HCMV antiviral assay: Antiviral assays for HCMV DNA were carried out byDNA hybridization as reported by Dankner, W. M., Scholl, D., Stanat, S.C., Martin, M., Souke, R. L. and Spector, S. A., J. Virol. Methods21:293-298, 1990. Briefly, subconfluent MRC-5 cells in 24-well culturedishes were pretreated for 24 h with various concentrations of drug inEagle s minimum essential medium (E-MEM) containing 2% FBS andantibiotics. The medium was removed and HCMV strains added aba dilutionthat will result in a 3-4+ cytopathic effect (CPE) in the no-drug wellsin 5 days. The virus was absorbed for 1′ h at 37° C., aspirated andreplaced with the drug dilutions. After 5 days of incubation HCMV DNAwas quantified in triplicate by nucleic acid hybridization using a CMVAntiviral Susceptibility Test Kit from Diagnostic Hybrids, Inc. (Athens,Ohio). The medium was removed and cells lysed according to themanufacturer s instructions. After absorption of the lysate, theHybriwix™ filters were hybridized overnight at 60° C. The Hybriwix™ werewashed for 30 min at 73° C. and counted in a gamma counter. The resultsare expressed as EC₅₀ (the 50% inhibitory concentration).

Preliminary experiments were performed on 1-O-hexadecylpropanediol (HDP)derivatives of cidofovir and adefovir, as shown in Table 1.

TABLE 1 Drug HCMV EC₅₀, μM CEM, CC₅₀, μM Selectivity Index CDV 0.45 ±0.09 (3) 857 1,900 cCDV 0.47 ± 0.13 (3) >1000 >2,100 HDP-cCDV 0.0005 (2)30 59,600 Adefovir    55 (1) HDP-Adefovir  0.01 (1) — —

As the results in Table 1 indicate, 1-O-hexadecylpTopanediol-3-cyclicCDV (HDP-cCDV) was >900 times more active than CDV or cyclic CDV. Whilemore cytotoxic, the selectivity index against HCMV in rapidly dividingcells was >59,000 vs. 1,900 to >2,100 for the underivatized CDV's. Basedon these promising preliminary results, further experiments were carriedout using additional invention compounds. These further experiments aredescribed as follows.

Cytotoxicity of Test Compounds In Vitro:

Subconfluent human lung fibroblast cells (MRC-5, American Type CultureCollection, Rockville, Md.) in 24-well plates were treated with drugsdiluted in E-MEM (Gibco BRL, Grand Island, N.Y.) supplemented with 2%fetal bovine serum and antibiotics. After 5 days of incubation at 37°C., the cell monolayer was visually inspected under magnification andthe concentration of drug which caused a 50% reduction in cell numberwas estimated.

The data obtained from these experiments is shown in Table 2.

TABLE 2 Inhibition of Human CMV Replication in MRC-5 Human LungFibroblasts Assayed by DNA Reduction Selectivity Compound EC₅₀, μM CC₅₀,μM Index Cidofovir (CDV) 0.46 >1000 >2174 Cyclic Cidofovir (cDCV)0.47 >1000 >2128 1-O-hexadecylpropanediol-3-CDV 2 × 10⁻⁶ 10 5 × 10⁶1-O-hexadecylpropanediol-3- 3 × 10⁻⁴ 320 1 × 10⁶ cCDV1-O-octadecylpropanediol-3-CDV 3 × 10⁻⁵ 32 1 × 10⁶1-O-octadecylpropanediol-3-cCDV 3 × 10⁻⁴ 320 1 × 10⁶1-O-octadecylethanediol-2-CDV >1 × 10⁻⁹  210  2 × 10¹¹1-O-octadecylethanediol-2-cCDV 3 × 10⁻⁴ 320 1 × 10⁶ Hexadecyl-cCDV 0.046.5 163 Adefovir (ADV) 55 >1000 >18 1-O-hexadecylpropanediol-3-ADV 0.106.5 65 1-O-octadecyl-sn-glycero-3-ADV 0.21 — — EC₅₀—50% effectiveconcentration; CC₅₀—50% cytotoxic concentration; selectivityindex—CC₅₀/EC₅₀. EC₅₀ results are the average of 3 to 6 determinations,with the exception that ADV is a single replication done in duplicate

As the results shown in Table 2 indicate, compounds of the invention areuniformly more active and selective than underivatized cidofovir, cycliccidofovir and adefovir.

Example 18 Effect of HDP-CCDV on Poxvirus Replication, In Vitro

The activity of cidofovir (CDV), cyclic cidofovir (cCDV), and1-O-hexadecylpropanediol-3-cCDV (HDP-cCDV) were tested for antiviralactivity in human foreskin fibroblasts infected with vaccinia virus orcowpox virus by measuring the dose dependent reduction in cytopathiceffect (CPE). Preliminary vaccinia and cowpox EC₅₀ values weredetermined in a CPE reduction assay in human foreskin fibroblast (HFF)cells. The data thus obtained is shown in Table 3.

TABLE 3 Vaccinia Cowpox, HFF Cells, Drug EC₅₀, μM EC50, μM CC50, μM CDV1.80 2.10 89.8 Cyclic CDV 0.97 0.72 >100 HDP-cCDV 0.11 <0.03 >100Control lipid >100 >100 >100

As shown in Table 3, HDP-cCDV was highly active against vaccinia viruswith an IC₅₀ value of 0.11 μM versus 0.97 and 1.8 μM for cCDV and CDV,respectively. In cowpox infected cells HDP-cCDV was extremely effectivewith an IC₅₀ of <0.03 μM versus 0.72 and 2.1 for cCDV and CDV,respectively. Based on this promising preliminary data, the effects ofinvention cidofovir analogs on the replication of other orthopox viruseswas investigated.

Poxvirus Antiviral Cytopathic Effect (CPE) Assay:

At each drug concentration, three wells containing Vero cells wereinfected with 1000 pfu/well of orthopoxvinis and three others remaineduninfected for toxicity determination. Plates were examined and stainedafter the virus-infected, untreated cells showed 4+ CPE. Neutral red wasadded to the medium and CPE was assessed by neutral red uptake at 540ran. The 50% inhibitory (EC50) and cytotoxic concentrations (CC50) weredetermined from plots of the dose response. The results are shown inTable 4.

TABLE 4 EC50, μM Variola Major, Variola Major, Variola Minor, CC₅₀Compound Vaccinia Cowpox Bangladesh Yamada Garcia μM CDV 2.2 3.8 100 —— >100 cCDV — — 100 — — >100 HDP- <0.03 <0.03 0.0015 0.0015 0.0006 >0.1CDV HDP- 0.11 <0.03 >0.01 — — >0.1 cCDV EC₅₀—50% effectiveconcentration; CC₅₀—50% cylotoxic concentration in Verocells;selectivity index - CC₅₀/EC₅₀; Abbreviations as in Table 2. Results arethe average of 3 determinations.

As shown in Table 4, invention compounds were substantially more activethan the underivatized CDV or cCDV against vaccinia, cowpox, and varioussmallpox strains.

Example 19 Effect of L-O-Hexadecyipropanediol-3-Adefovir (HDP-ADV) onHIV-1 Replication, In Vivo

Preliminary experiments in the inhibition of HTV-1 replication byinvention compounds were performed as follows. Drug assays were carriedout as previously described by Larder et. al., Antimicrobial Agents &Chemotherapy, 34:436-441, 1990. HIV-1_(LA1) infected HT4-6C cells wereexposed to drugs as indicated and incubated for 3 days at 37° C. Thecells were fixed with crystal violet to visualize plaques. Antiviralactivity was assessed as the percentage of control plaques (no drug)measured in drug treated samples. The EC₅₀ is the micromolarconcentration which reduces plaque number by 50%. The activity ofadefovir was compared with AZT (zidovudine) and1-O-hexadecylpropanediol-3-adefovir (HDP-ADV) in HIV-1 infected HT4-6Ccells. The results are shown in Table 5.

TABLE 5 Drug EC50, μM, in HIV-1 plaque reduction assay AZT 0.007Adefovir 16.0 HDP-ADV 0.0001

Adefovir was moderately active with an EC₅₀ of 16 μM. AZT was highlyactive as anticipated (EC₅₀ 0.007 μM) but HDP-ADV was the most active ofthe three compounds with an EC₅₀ of 0.0001 μM, more than 5 logs moreactive than adefovir itself Based on these promising preliminaryresults, father experiments were carried out as follows.

HIV-1 antiviral assay: The effect of antiviral compounds on HIVreplication in CD4-expressing HeLa HT4-6C cells, was measured by aplaque reduction assay (Larder, B. A., Chesebro, B. and Richman, D. D.Antimirob. Agents Chemother., 34:436-441, 1990). Briefly, monolayers ofHT4-6C cells were infected with 100-300 plaque forming units (PFU) ofvirus per well in 24-well microdilution plates. Various concentrationsof drug were added to the culture medium, Dulbecco's modified Eaglemedium containing 5% FBS and antibiotics, as noted above. After 3 daysat 37° C., the monolayers were fixed with 10% formaldehyde solution inphosphate-buffered saline (PBS) and stained with 0.25% crystal violet tovisualize virus plaques. Antiviral activity was assessed as thepercentage of control plaques measured in drug-treated samples.Cytotoxicity was assessed by the method of Hostetler et al., AntiviralResearch, 31:59-67, 1996. The results are shown in Table 6.

TABLE 6 Inhibition of HIV Replication in HT4-6C Cells by PlaqueReduction Compound EC₅₀, μM CC₅₀, μM Selectivity Index Adefovir (ADV)8.2 >1000 >122 1-O-hexadecylpropanediol-3- 0.008 6.5 813 ADV EC₅₀—50%effective concentration; CC₅₀—50% cyfotoxic concentration; selectivityindex—CC₅₀/EC₅₀. EC₅₀ values are the average of 4 experiments.

As the results in Table 6 readily indicate, invention compound1-O-hexadecylpropanediol-3-ADV is more active and selective thanadefovir.

Example 20 Effect of Cidofovir Analogs on Herpes Virus Replication

HSV-1 Antiviral Assay:

Subconfluent MRC-5 cells in 24-well culture dishes were inoculated byremoving the medium and adding HSV-1 virus at a dilution that willresult in a 3-4+ CPE in the no-drug well in 20-24 h. This was absorbedfor 1 h at 37° C., aspirated and replaced with various concentrations ofdrugs in E-MEM containing 2% FBS and antibiotics. After approximately 24h of incubation, HSV DNA was quantified in triplicate by nucleic acidhybridization using a HSV Antiviral Susceptibility Test Kit fromDiagnostic Hybrids, Inc. (Athens, Ohio). The medium was removed andcells lysed according to the manufacturer s instructions. Afterabsorption of the lysate, the Hybriwix™ filters were hybridizedovernight at 60° C. The Hybriwix were washed for 30 min at 73° C. andcounted in a gamma counter. Cytotoxicity was assessed as described inExample 17. EC₅₀ and CC₅₀ values thus obtained are shown in Table 7.

TABLE 7 Inhibition of Human HSV Replication in MRC-5 Human LungFibroblasts Assayed by DNA Reduction Selectivity Compound EC₅₀, μM CC₅₀,μM Index Cidofovir (CDV) 1.20 >1000 >800 Cyclic Cidofovir (cCDV)2.10 >1000 >475 1-O-hexadecylpropanediol-3-CDV 4 × 10⁻⁷ 10 25 10⁶1-O-hexadecylpropanediol-3- 0.030 320 10,667 cCDV1-O-octadecylpropanediol-3-CDV 0.003 32 10,6671-O-octadecylpropanediol-3-cCDV 0.330 320 9701-O-octadecylpropanediol-2-CDV 0.002 210 105,0001-O-octadecylpropanediol-2-cCDV 0.008 320 40,000 Abbreviations as inTable 2. EC₅₀—50% effective concentration; CC₅₀—50% cytotoxicconcentration; selectivity index—CC₅₀/EC₅₀. EC₅₀ values are the averageof two experiments with the exception of HDP-CDV which is a singledetermination in duplicate.

As shown in Table 7, all invention compounds are more active againstHSV-1 than the underivatized nucleotide phosphonates, cidofovir orcyclic cidofovir.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those of ordinary skill in the artin light of the teaching of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the claims.

What is claimed is:
 1. A compound of the formula:

wherein R¹ is hydrogen, —CH₃, —CH₂OH, —CH₂F, —CH═CH₂, or —CH₂N₃; B iscytosin-1-yl; and R⁸ is alkylglycerol, 1-S-alkylthioglycerol, oralkoxyalkanol.
 2. The compound of claim 1, wherein R⁸ is alkylglycerol.3. The compound of claim 1, wherein R⁸ is 1-S-alkylthioglycerol.
 4. Thecompound of claim 1, wherein R⁸ is alkoxyalkanol.
 5. The compound ofclaim 4, wherein R⁸ is alkoxyethanol.
 6. The compound of claim 4,wherein R⁸ is alkoxypropanol.
 7. The compound of claim 6, wherein R⁸ ishexadecyloxypropanol.
 8. The compound of claim 6, wherein R⁸ isoctadecyloxypropanol.
 9. The compound of claim 1, wherein R¹ is —CH₂OH.10. The compound of claim 9, wherein R⁸ is alkylglycerol.
 11. Thecompound of claim 9, wherein R⁸ is 1-S-alkylthioglycerol.
 12. Thecompound of claim 9, wherein R⁸ is alkoxyalkanol.
 13. The compound ofclaim 12, wherein R⁸ is alkoxyethanol.
 14. The compound of claim 12,wherein R⁸ is alkoxypropanol.
 15. The compound of claim 14, wherein R⁸is hexadecyloxypropanol.
 16. The compound of claim 14, wherein R⁸ isoctadecyloxypropanol.
 17. A pharmaceutical composition comprising aneffective amount of the compound of claim 1 and a pharmaceuticallyacceptable carrier.
 18. The pharmaceutical composition of claim 17,wherein the composition is formulated as a solid dosage form.
 19. Thepharmaceutical composition of claim 17, wherein the composition isformulated as a capsule, tablet, aerosol, solution, or suspension, or isa topical formulation or a solution.